Upfront Screening by Quantitative Real-Time PCR Assay Identifies NUP98::NSD1 Fusion Transcript in Indian AML Patients
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. Plasmid Construction
2.3. Designing of Primers and Probes
2.4. Sensitivity of Assay
2.5. Specificity of the Assay
2.6. RNA Extraction and cDNA Preparation
2.7. Quantitative qRT-PCR
2.8. Statistical Data Analysis
2.9. Sanger’s Sequencing for Validation of NUP98-NSD1 Fusion
2.10. FLT3-ITD Fragment Analysis
3. Results
3.1. Development of a qRT-PCR Assay to Determine NUP98::NSD1 Fusion Transcript
3.2. Screening of Patients for the Presence of NUP98::NSD1 Fusion in the Indian Cohort
3.3. Sanger Sequencing Confirmation of NUP98::NSD1 Breakpoint Fusion
3.4. FLT3-ITD Status in NUP98::NSD1 Fusion Positive Patients
3.5. NUP98::NSD1 Fusion Patients Have Altered the Expression of Genes Associated with Self-Renewal and Differentiation
3.6. NUP98::NSD1 Patients Show Poor Responses to Induction Therapy
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Cloning Primers | |
---|---|
NUP98::NSD1 fusion forward | 5′AAAGATCATGACATAGATTACAAGGATGACGATGA CAAGGCCATGTTTAACAAATCATTTGGAACACC-3′ |
NUP98::NSD1 fusion reverse | 5′AGTCGAGGCTGATCAGCGGGTTTAAACGGGCC CTCTAGACCTACTTCTGTTCTGATTCTGCACACTT-3′ |
Sequencing Primers | |
NUP98-Seq F | 5′-ACTCTTGGAACTGGGCTTGG-3′ |
NSD1-Seq R | 5′-GGCTAGAAGGCTTTCCTCTTC-3′ |
qRT-PCR primers | |
NUP98 t(5,11) F | 5′-GGCCCCTGGATTTAATACTACGA-3′ |
NSD1 t(5,11)R | 5′-CTTCCTAAGGCGTTTCTTCTCTGA-3′ |
NUP98-NSD1 t(5,11) probe | 5′-FAM-TTTGGAGCCCCCCAGGCC-MGB NFQ-3′ |
ABL1-F | 5′-CCCAGAGAAGGTCTATGAACTCATG-3′ |
ABL1-R | 5′-AGGAGGGCCGGTCAGA-3′ |
ABL1 probe | 5′-VIC-TCCACTGCCAACATGC-MGB NFQ-3′ |
HOXA1 F | 5′-CCCTCGGACCATAGGATTACAA-3′ |
HOXA1 R | 5′-GCCGCCGCAACTGTTG-3′ |
HOXA3 F | 5′-CGACAGCTCGGCGATCTAC-3′ |
HOXA3 R | 5′-CGGGTACGGCTGCTGATT-3′ |
HOXA4 F | 5′-GGTGGTGTACCCCTGGATGA-3′ |
HOXA4 R | 5′-GACTTGCTGCCGGGTATAGG-3′ |
HOXA5 F | 5′-GGAGTTCCACTTCAACCGTTACC-3′ |
HOXA5 R | 5′-CGGAGAGGCAAAGAGCATGT-3′ |
HOXA6 F | 5′-GTCTGGTAGCGCGTGTAGGT-3′ |
HOXA6 R | 5′-CCCTGTTTACCCCTGGATG-3′ |
HOXA7 F | 5′-CTTCTCCAGTTCCAGCGTCT-3′ |
HOXA7 R | 5′-AAGCCAGTTTCCGCATCTAC-3′ |
HOXA9 F | 5′-CCACGCTTGACACTCACACT-3′ |
HOXA9 R | 5′-GCTCTCATTCTCGGCATTGT-3′ |
HOXA10 F | 5′-TCTTTGCTGTGAGCCAGTTG-3′ |
HOXA10 R | 5′-CTCCAGCCCCTTCAGAAAAC-3′ |
HOXA11 F | 5′-CGGCCACACTGAGGACAAG-3′ |
HOXA11 R | 5′-AACTCTCGCTCCAGCTCTCG-3′ |
HOXB6 F | 5′-TCCCCTCCCAATGAGTTCCT-3′ |
HOXB6 R | 5′-ACTCCTGCCCGCTGGC-3′ |
PRDM16 F | 5′-TGCCGCACGCAGATCA-3′ |
PRDM16 R | 5′-GGGAGGAGGTAGTGCTGAACAT-3′ |
MECOM F | 5′-CGGAGTGTGGCAAAACGTT-3′ |
MECOM R | 5′-GCTGTGGATGTGCTTGTGTTGT-3′ |
NKX2-3 F | 5′-GGTTCCAGAATCGCAGGTACAA-3′ |
NKX2-3 R | 5′-GCGCCAAGCTCCAGAGACT-3′ |
VENTX F | 5′-GGCTGGCCAGGGAGATG-3′ |
VENTX R | 5′-TGCGGCGATTCTGAAACC-3′ |
UTF F | 5′-GACCAGCTGCTGACCTTGAA-3′ |
UTF R | 5′-CTGCCCAGAATGAAGCCCA-3′ |
ASL F | 5′-CAGCATGGATGCCACTAGTGA-3′ |
ASL R | 5′-CACAGCGAAGCCCAGAACA-3′ |
GAPDH F | 5′-AATCCCATCACCATCTTCCA-3′ |
GAPDH R | 5′-TGGACTCCACGACGTACTCA-3′ |
Fragment analysis primers | |
FLT3 exon14 F | 5′-FAM-AGCAATTTAGGTATGAAAGCCAGCTA-3′ |
FLT3 exon14 R | 5′-CTTTCAGCATTTTGACGGCAACC-3′ |
Patient No. | Age (in Years) | Sex | FAB | WBC Count at Diagnosis (WBC Count /L) | BM Blast (%) | FLT3-ITD Status | Treatment Protocol | Induction Chemo Response | HSCT | Outcome |
---|---|---|---|---|---|---|---|---|---|---|
Adult #1 | 57 | M | M4 | 19.2 * 109 | 70% | FLT3-ITD Positive (AR 0.35) | 3 + 7 f/b HAM | Induction failure | No | Expired due to severe sepsis & respiratory failure |
Adult #2 | 64 | F | M2 | 12.8 * 109 | 95% | FLT3-ITD Positive (AR 0.35) | Azacitidine + Venetoclax f/b HAM | Induction failure | No | Died due to disease progression |
Adult #3 | 33 | M | M3 | 20 * 109 | 95% | FLT3-ITD Positive (AR 0.31) | 3 + 7 + Midostaurin | Induction failure | No | Died due to intracranial bleed. |
Pediatric #1 | 15 | M | M2 | 19.9 * 109 | 55% | FLT3-ITD Positive (AR 0.36) | 3 + 7 + Midostaurin | Achieved remission | Yes | Disease relapsed 7 months after allogenic stem cell transplant |
Pediatric #2 | 15 | F | M2 | 18.6 * 109 | 71% | FLT3-ITD Positive (AR 0.21) | 3 + 7 + Midostaurin | Induction failure | No | Expired due to MDR sepsis (kliebsella pneumoniae) |
Pediatric #3 | 12 | F | M1 | 17.9 * 109 | 90% | FLT3-ITD Positive (AR 0.37) | 3 + 7 + Midostaurin f/b HAM+ Midostaurin | Induction failure | Yes | Disease relapsed 4 months after allogenic stem cell transplant |
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Shah, A.; Sharma, A.; Katiyar, S.; Gupta, A.; Chaturvedi, C.P. Upfront Screening by Quantitative Real-Time PCR Assay Identifies NUP98::NSD1 Fusion Transcript in Indian AML Patients. Diagnostics 2022, 12, 3001. https://doi.org/10.3390/diagnostics12123001
Shah A, Sharma A, Katiyar S, Gupta A, Chaturvedi CP. Upfront Screening by Quantitative Real-Time PCR Assay Identifies NUP98::NSD1 Fusion Transcript in Indian AML Patients. Diagnostics. 2022; 12(12):3001. https://doi.org/10.3390/diagnostics12123001
Chicago/Turabian StyleShah, Arunim, Akhilesh Sharma, Shobhita Katiyar, Anshul Gupta, and Chandra Prakash Chaturvedi. 2022. "Upfront Screening by Quantitative Real-Time PCR Assay Identifies NUP98::NSD1 Fusion Transcript in Indian AML Patients" Diagnostics 12, no. 12: 3001. https://doi.org/10.3390/diagnostics12123001
APA StyleShah, A., Sharma, A., Katiyar, S., Gupta, A., & Chaturvedi, C. P. (2022). Upfront Screening by Quantitative Real-Time PCR Assay Identifies NUP98::NSD1 Fusion Transcript in Indian AML Patients. Diagnostics, 12(12), 3001. https://doi.org/10.3390/diagnostics12123001